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Uridine uptake by nerve cells of the grivet monkey and its relation to cytoplasmic ribonucleic acid concentration
Neurochemistry International Vol. 5, No. 5, pp. 553-557, 1983
01974)186/83 $3.00 + 0.00 © 1983 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
URIDINE UPTAKE BY NERVE CELLS OF THE GRIVET MONKEY AND ITS RELATION TO CYTOPLASMIC RIBONUCLEIC ACID CONCENTRATION H. PAKKENBERG a n d R. FOG Laboratory of Neurology, Hvidovre University Hospital, DK-2650 Hvidovre, Denmark
(Received 23 October 1982; accepted 4 January 1983) Abstract--Following intravenous injection of tritiated uridine to a grivet monkey, the uptake in nerve cell nuclei was determined autoradiographically in different brain regions, in the choroid plexus, liver and kidney. It is shown (1) that there are obvious differences in the labelling in different brain areas, and (2) that the labelling, compared with earlier results in mice, is the same in some regions but different in others. The uridine labelling was inversely related to the microspectrophotometrically determined cytoplasmic concentration of ribonucleic acid. This might indicate that cells with a high uridine count and a low cytoplasmic ribonucleic acid concentration have a high metabolic activity.
EXPERIMENTAL PROCEDURES
In the last few years there has been a spectacular accumulation of knowledge on regional metabolic activity of the brain. The oxygen uptake (Siesjr, 1977), the glucose metabolism (Sokoloff, 1981 ; Reivich et al., 1975), as well as processes involving several other substances (Gervas-Camacho, Baljagon a n d De Feudis, 1970) have been studied. Along the same lines we found considerable variations in the uridine uptake in different areas of the mouse brain (Fog and Pakkenberg, 1976). It may be erroneous to extrapolate observations in small rodents to what takes place in primates because metabolism varies from animal species to animal species (Juorio, 1973). In this study we attempted to bridge the species variation by investigating regional 3H-uridine uptake in a grivet monkey a n d compare the results to the findings in mice. A n o t h e r aim of the investigation was to study the relation between the nerve cell concentration of cytoplasmic R N A a n d its metabolic activity. M o s t investigators have found an increased RNA concentration with increased nerve cell function (Pevzner, 1965, 1971; Kernell a n d Peterson, 1970; Peterson a n d Kernell, 1970). Others observed a n inverse relationship (Orrego, 1967; G e i n i s m a n n , 1971; P a k k e n b e r g a n d T h o m s e n , 1964), or n o n e at all ( B o c h a r o v a et al., 1972), depending on the stimulation time. W e investigated quantitatively the cytoplasmic degree of basophilia, and determined autoradiographically the uptake of tritiated uridine in the same cells as an expression of RNA synthesis.
A monkey of the species group Cercopithecus aethiops from Ethiopia, weight 3.7 kg, was injected with 30 mCi of 5-3H-uridine intravenously (sp. act. 29.7 mCi (1.10 TBq)/mmol). One hour later the animal was sacrificed by intramuscular injection of Ketalar 40 mg followed by mebumal 6~o, 1.8 ml intracardially. Immediately thereafter it was perfused first with sodium chloride 0.9~o, 300ml through the left ventricle after opening the right atrium and then with 4~o formalin. To avoid artefacts from shrinking of the cells, the brain was left in situ for 24 h. It was then removed, further fixed in formalin for 8 days, cut into blocks, which were rinsed in water, dehydrated in alcohol, cleared in chloroform, embedded in paraffin and cut in 4 pm sections. The sections were dipped in Ilford Nuclear Research Emulsion K-5, exposed at 4°C for 2 weeks and developed in Amidol for 4 min at 18°C. The sections were stained with haematoxylin-eosin, and the grains in 25 nuclei counted independently by both authors. The nucleus diameter was determined with an ocular micrometer and the grain number corrected to a diameter of 12/~m in relation to nuclei area. Another series of sections was treated in the same way, except that they were stained with gallocyanin-chromealum, pH 1.68, which specifically stain RNA in the cytoplasm. The thickness of the sections was determined by focusing on the upper and lower surface with high magnification. Only sections with a thickness of 4 + 0.2/~m were used. As in the first series, the number of grains in 25 nuclei of the cerebral cortex was counted. The diameters of the nuclei were measured and the number of grains was corrected for differences in nucleus size as described above. The extinction value was measured microspectrophotometrically at 2 points of the cytoplasm. The diameter of the field of measurement in the microspectrophotometer was 1.6/tin. In some locations, the rim of the cytoplasm was 553
554
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+
-I-
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~
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Frontal I(~e 0
Tempoea{ lobe
F'clrleto{ lobe
Occ ~ollat lobe
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in the nuclei in the 2 regions are ahnost identical. On the other hand, the nuclear diameter of pyramidal cells in the hippocampus and the thalamus are ver? similar, but gram counts are significantly lower in the hippocampus than in the thalamus. Uridine uptake is quite similar in all parts of the cortex, although slightly increased grain counts are noted in the third and fifth layers. In Table 1 we have compared the grain counts fiom this investigation on the monkey with earlier investigations on mice (Fog and Pakkenberg, 1976). The numbers are c o m p a r a b l e because of a similar exposure time and nucleus area. The monke}, however. was injected with a 200o lower dose of uridine per kg. Striking differences are observed in the second layer of the cortex, the Purkinje cells and the hippocampus cells, all other counts being almost identical. In Table 2 considerable differences in RNA concentration in the cytoplasm in the different brain regions are demonstrated. In each region the extinction value of the different cells has been compared with the grain count of the cell nucleus. From Table 2 it is seen that the correlation coefficient in all cases is negative, and in 8 out of 12 cases significantly different from zero. This indicates that the highest grain counts have been found in cells with a low RNA concentration.
J
Fig. 1. The number of grains in nerve ccll nuclei in the different regions of a grivet brain and in liver and kidney are shown l h after H3-5-uridine injection, the standard error is indicated at the top of each column. too narrow for extinction measurements to be made, e.g. in the dentate gyrus. In the cortical sections, only the nerve cells in the fifth layer were measured. RESULTS As illustrated in Fig. l, the uridine uptake varies from location to location in the brain. The grain counts are given per area, and are therefore comparable from region to region. It is noteworthy that there is no correlation between grain counts and the nuclear diameters in different regions. The diameter of the nuclei in the corpus striatum, e.g. is 11.5 # m as against 18.9 l~m in the cervical cord. The grain counts
DISCUSSION
Regional uridine uptake It is well-established that uridine is especially incorporated in RNA in the cell nucleus (Guroff, Hogans and Udenfriend, 1968). By the autoradiographical method, the small, easily soluble molecules are removed from the tissues by the histological procedure. Only large molecules are fixed. There is no direct correlation between the uridine uptake and the synthesis in the cells (Dunn and Bondy, 1974). However, it is reasonable to assume that the uridine uptake expresses the RNA synthesis, if the animal behaves
Table 1. A comparison between grains covering nerve cell nuclei in the mouse (Fog and Pakkenberg, 1976} and the grivet (grains/'100 jtm 2)
Cortex. 2nd layer Cortex, 5th layer Purkinje cells Striatum Hippocampus, pyr. layer Hippocampus, dentate gyrus Thalamus S.E. in brackets.
Mouse
Monkey
24.4 ( + 2.05) 10.8 ( ± 1.10t 22.0 ( + 3.181 11.4 ( ± 1.581 17.6 ( + 2.18t 7.0 (±0.98) 15.8 ( ± 1.491
9.8 (±0.55) 12.1 (_+0.49) 9.4 ( ± 0.501 I 1.6 ( +0.501 8.6 ( ± 0.401 8.1 ( +_0.53t 16.0 ( ± 0.511
Uridine uptake by nerve cells of the grivet monkey
555
Table 2. The extinction values of the nerve cell cytoplasm in 25 cells in different areas of a grivet brain; staining method: gallocyanine--chrome alum Extinction Frontal cortex Parietal cortex Temporal cortex Occipital cortex Striatum Thalamus Hippocampus (pyr. layer) Substantia nigra Facial nucleus Purkinje cells Spinal cord cervical Spinal cord lumbar
0.32 0.36 0.27 0.30 0.30 0.23 0.34 0.22 0.34 0.38 0.44 0.30
Range 0.22-0.42 0.28-0.46 0.21-0.36 0.18 0.40 0.22 0.39 0.15--0.36 0.23 0.43 0.10-0.39 0.19 0.54 0.28 0 . 5 0 0.3~0.55 0.21 -0.42
Correlation coefficient -0.22 - 0.02 -0.81"** -0.70*** -0.38 - 0.46* -0.67** -0.44* -0.59* -0.83*** -0.05 -0.44*
The correlation coefficients show the relation between uridine labelling and extinction values; the difference from zero is shown. * P < 0.05. ** P < 0.01. *** P < 0.001. normally during the experimental period (Rainbow, 1979). Thus, it is possible to obtain an expression of the metabolic activity (RNA synthesis) in a short period of time. The labelling of cell nuclei in mice is maximal ½h after uridine injection and remains unchanged during the following 6-12 h (Pakkenberg and Fog, 1972). Uridine labelling of RNA will label all RNA species such as ribosomal, transfer and messenger RNA. However, the amount of ribosomal and transfer RNA is rather stable and the estimation that messenger RNA is the principal class of RNA the metabolism of which is fast and can therefore be followed by the technique employed, appears probable. This assumption is supported by the observation, that the turnover time of ribosomal RNA is about 12 days (Von Hungen et al., 1968), but only 30min to 2 h for messenger RNA (Kimberlin, 1967). Our present results indicate that important regional differences in RNA synthesis exist in the normal grivet. Furthermore, in some regions (hippocampus, cerebellum and the second layer of the cortex) differences exist between grivet and mice (Fog and Pakkenberg, 1976). Thus it is confirmed that it is not possible to extrapolate conclusions concerning the uridine uptake (RNA synthesis) from one animal species to another. In order to investigate whether the uridine uptake is related to morphological, biochemical or physiological differences in the different brain areas, we compared our uridine findings with the capillary density in different brain areas (Campbell, 1939). No corr~c~ 5 / 5
D
relation was found. The protein concentration in the cat brain varies only very little from region to region (Gervas-Camacho, Baljagon and De Feudis, 1970). The variation of leucine uptake in the different brain areas in rats is quite different to that of uridine uptake (Altman, 1963). The variations of glucose uptake and cerebral blood flow are similar from region to region, but are different to the uridine uptake pattern (Schwartz and Sharp, 1978; Sokoloff, 1981); it has thus not been possible to find correlations between uridine uptake and other relevant parameters. Uridine labelling and cytoplasmic R N A concentration
Observations on the relationship between cell activity and cytoplasmic RNA concentration are conflicting. For example, Pevzner (1965), Peterson and Erulkar (1973) and Peterson and Kernell (1970) noted a relation between increased cytoplasmic RNA concentration and increased cell activity. On the other hand Bocharova et al. (1972) found an initial decrease in cytoplasmic R N A concentration with an increase after 40 and 7 0 m i n (150-200~o). In vitro investigations (Orrego, 1967) demonstrated a 4 0 ~ decrease in R N A synthesis after electrical stimulation; in contrast, Edstr6m and G r a m p p (1965) and G r a m p p and Edstr6m (1963) claimed that electric stimulation had no bearing on the R N A concentration. In patients who died in status epilepticus, a severe decrease of the R N A concentration in the cytoplasms has been found (Einarson and Krogh, 1955). Electrically induced convulsions (Hartelius,
556
H. PAKKENBERGand R. FoG
1952) in cats have induced foci o f c h r o m o p h o b i c cells (cells with a low cytoplasmic R N A concentration) in the cerebral cortex. The relation between cytoplasmic basophilia and uridine uptake in the nucleus was investigated by Koenig (1958). He found that chromophobic nerve cells incorporate more RNA precursor than chromophilic neurons. Engel and Morrell (1970) observed no correlation between the degree o f chromophilia and the grain counts in normal neurons. We have demonstrated a relation between low RNA concentration in the cytoplasm and high uridine labelling of the nucleus. This might be an expression of an increased activity of these neurons. Earlier we showed that the number of grains in the cytoplasm increases about 6 h after the uridine injection (Pakkenberg and Fog, 1972), but an investigation of the uridine labelling in the nucleus and a quantitative determination of RNA concentration in the cytoplasm in the same cell, has not been previously made. The grain counts in the choroid plexus are of the same order of magnitude as in the most active regions in the brain. In the mouse (Pakkenberg and Fog, 1977) we found a much higher uptake in the plexus than in the nerve cells in the cortex. The relatively modest uptake in the hepatocytes is striking. In the mouse the uptake in the liver is 100~o higher than in the nerve cells (Pakkenberg and Fog, 1977). We have no explanation of these differences.
REFERENCES Altman, J. (1963). Differences in the utilization of tritiated leucine by single neurones in normal and exercised rats. Nature, Lond. 199, 777 780. Bocharova, L. S., Borovyagin, V. L., Dyakonova, T. L., Warton, S. S. and Veprintslev, B. N. (1972). Ultrastructure and RNA synthesis in a molluscan giant neuron under electrical stimulation. Brain Res. 36, 371 384. Campbell, A. (1939). Variation in vascularity and oxidase content in different regions of the brain of the cat. Arch. Neurol. Psychiat. 41,223 242. Dunn, A. and Bondy, S. (1974). Functional Chemistry o] the Brain, pp. 208 209. Spectrum, New York. Edstr~Sm, J.-E. and Grampp, W. (1965). Nervous activity and metabolism of ribonucleic acids in the crustacean stretch receptor neuron. J. Neurochem. 12, 735 741. Einarson, L. and Krogh, E. (1955). Variations in the basophilia of nerve cells associated with increased cell activity and functional stress. J. Neurol. Neurosurg. Psychiat, t8, l 11. Engel, J. Jr and Morrell, F. (1970). Turnover of RNA in normal and secondarily epileptogenic rabbit cortex. Exp. Neurol. 26, 221 238. Fog, R. and Pakkenberg, H. (1976). Relative uptake of tritiated uridine in various brain areas in the mouse. J. Neurochem. 2"7, 1261 1262.
Geinismann, Yu.Ya. (19711. Nucleic acid content of spinal motoneurones and their satellites under orthodromic and antidromic stimulation. A cytospectrophotometric study. Brain Res. 28, 251 262. Gervas-Camacho, J., Baljagon, G. and De Feudis, F. (1970). Constancia del contenido total de proteinas en varias regiones del sistema nervioso central del gato. Rev. Espanola, Fiziol. 32, 111-114. Grampp, W. and Edstr6m, J.-E. (1963). The effect of nervous activity on ribonucleic acid of the crustacean receptor neuron. J. Neurochem. 10, 725 731. Guroff, G., Hogans, A. F. and Udenfriend, S. (1968). Biosynthesis of ribonucleic acid in rat brain slices. J. Neurochem. 15, 489 497. Hartelius, H. (1952). Cerebral changes following electrically induced convulsions. Acta Psyeh. Neut. Scand.~ suppL 77. Juorio, A. V. (1973). The distribution of catecholamines in the hypothalamus and other brain areas of some lower vertebrates. J. Neurochem. 20, 641 645. Kernell, D. and Peterson, R. (1970). The effect of spike activity versus synaptic activation on the metabolism of ribonucleic acid in a molluscan giant neurone. J. Neurochem. 17, 1087 1094. Kimberlin, R. (1967). RNA synthesis in mouse brain. J. Neuroehem. 14, t23 134. Koenig, H. (1958). An autoradiographic study of thc nucleic acid and protein turnover in the mammalian neuraxis. J. hiophvs, hiochem. CytoL 4, 785 792. Siesj6, B. K. 11977). Physiological aspects of brain energy metabolism. In Biochemical Correlates ~[" Brain Structure and Function. (Davidson A. N. ed.) pp. 175 213. Academic Press, New York. Orrego, F. (1967). Synthesis of RNA in normal and electrically stimulated brain cortex slices m vitro. J. Neurochem. 14, 851 858. Pakkenberg, H. and Fog, R. (1972). Kinetics of 3H-5oridine incorporation in brain cells of the mouse. Exp. Neurol. 36, 405 410. Pakkenberg, H. and Fog, R. (1977). Autoradiographic study of nucleic acid precursor incorporation in nerve cells of the mouse brain. Neuroseienee 2, 1077 1084. Pakkenberg, H. and Thomsen, E. (1964). Cytoplasmic basophilia in spiral ganglion cells of the Guinea-pig following strong acoustic stimulation. Acta oto-laryng. 58, 300 311. Peterson, R. P. and Erulkar, S. D. (1973). Parameters of stimulation of RNA synthesis and characterization by hybridization in a molluscan neuron. Brain Res. 60, 177 190. Peterson, R. P. and Kernell, D. (19701. Effects of nerve stimulation on the metabolism of ribonucleic acid in a molluscan giant neurone. J. Neurochem. 17, 1075 1085. Pevzner, L. Z. (1965). Topochemical aspects of nucleic acid and protein metabolism within the neuron-neuroglia unit of the superior cervical ganglion. J. Neurochem. 12, 993 1002. Pevzner, L. Z. (1971). Topochemicat aspects of nucleic acid and protein metabolism within the neuron-neurglia unit of the spinal cord anterior horn. J. Neurochem. 18, 895 907. Rainbow, T. C. (1979). Role of RNA and protein synthesis in memory formation. Neurochem. Res. 4, 298-312. Reivich, M., Sokoloff, L., Kennedy, C. and Das Rosiers, M. (1975~. An autoradiographic method for the measurement of local glucose metabolism in the brain. In Brain
Uridine uptake by nerve cells of the grivet monkey Work (Ingvar D. H. and Lassen N. A. eds) pp. 377 384. Munksgaard, Copenhagen. Schwartz W. and Sharp, F. (1978). Autoradiographic maps of regional brain glucose consumption in resting, awake rats using 14C 2-deoxyglucose. J. comp. Neurol. 177, 335-360. Sokoloff, L. (1981). The deoxyglucose method for the
557
measurement of local glucose utilisation and measuring of functional activity in the central nervous system. Int. Rev. Neurobiol. 22, 287 333. Von Hungen, K., Mahler, H. and Moore, W. (1968). Turnover of protein and ribonucleic acid in synaptic subcellular fractions from rat brain. J. biol. Chem. 243, 1415-1423.
01974)186/83 $3.00 + 0.00 © 1983 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
URIDINE UPTAKE BY NERVE CELLS OF THE GRIVET MONKEY AND ITS RELATION TO CYTOPLASMIC RIBONUCLEIC ACID CONCENTRATION H. PAKKENBERG a n d R. FOG Laboratory of Neurology, Hvidovre University Hospital, DK-2650 Hvidovre, Denmark
(Received 23 October 1982; accepted 4 January 1983) Abstract--Following intravenous injection of tritiated uridine to a grivet monkey, the uptake in nerve cell nuclei was determined autoradiographically in different brain regions, in the choroid plexus, liver and kidney. It is shown (1) that there are obvious differences in the labelling in different brain areas, and (2) that the labelling, compared with earlier results in mice, is the same in some regions but different in others. The uridine labelling was inversely related to the microspectrophotometrically determined cytoplasmic concentration of ribonucleic acid. This might indicate that cells with a high uridine count and a low cytoplasmic ribonucleic acid concentration have a high metabolic activity.
EXPERIMENTAL PROCEDURES
In the last few years there has been a spectacular accumulation of knowledge on regional metabolic activity of the brain. The oxygen uptake (Siesjr, 1977), the glucose metabolism (Sokoloff, 1981 ; Reivich et al., 1975), as well as processes involving several other substances (Gervas-Camacho, Baljagon a n d De Feudis, 1970) have been studied. Along the same lines we found considerable variations in the uridine uptake in different areas of the mouse brain (Fog and Pakkenberg, 1976). It may be erroneous to extrapolate observations in small rodents to what takes place in primates because metabolism varies from animal species to animal species (Juorio, 1973). In this study we attempted to bridge the species variation by investigating regional 3H-uridine uptake in a grivet monkey a n d compare the results to the findings in mice. A n o t h e r aim of the investigation was to study the relation between the nerve cell concentration of cytoplasmic R N A a n d its metabolic activity. M o s t investigators have found an increased RNA concentration with increased nerve cell function (Pevzner, 1965, 1971; Kernell a n d Peterson, 1970; Peterson a n d Kernell, 1970). Others observed a n inverse relationship (Orrego, 1967; G e i n i s m a n n , 1971; P a k k e n b e r g a n d T h o m s e n , 1964), or n o n e at all ( B o c h a r o v a et al., 1972), depending on the stimulation time. W e investigated quantitatively the cytoplasmic degree of basophilia, and determined autoradiographically the uptake of tritiated uridine in the same cells as an expression of RNA synthesis.
A monkey of the species group Cercopithecus aethiops from Ethiopia, weight 3.7 kg, was injected with 30 mCi of 5-3H-uridine intravenously (sp. act. 29.7 mCi (1.10 TBq)/mmol). One hour later the animal was sacrificed by intramuscular injection of Ketalar 40 mg followed by mebumal 6~o, 1.8 ml intracardially. Immediately thereafter it was perfused first with sodium chloride 0.9~o, 300ml through the left ventricle after opening the right atrium and then with 4~o formalin. To avoid artefacts from shrinking of the cells, the brain was left in situ for 24 h. It was then removed, further fixed in formalin for 8 days, cut into blocks, which were rinsed in water, dehydrated in alcohol, cleared in chloroform, embedded in paraffin and cut in 4 pm sections. The sections were dipped in Ilford Nuclear Research Emulsion K-5, exposed at 4°C for 2 weeks and developed in Amidol for 4 min at 18°C. The sections were stained with haematoxylin-eosin, and the grains in 25 nuclei counted independently by both authors. The nucleus diameter was determined with an ocular micrometer and the grain number corrected to a diameter of 12/~m in relation to nuclei area. Another series of sections was treated in the same way, except that they were stained with gallocyanin-chromealum, pH 1.68, which specifically stain RNA in the cytoplasm. The thickness of the sections was determined by focusing on the upper and lower surface with high magnification. Only sections with a thickness of 4 + 0.2/~m were used. As in the first series, the number of grains in 25 nuclei of the cerebral cortex was counted. The diameters of the nuclei were measured and the number of grains was corrected for differences in nucleus size as described above. The extinction value was measured microspectrophotometrically at 2 points of the cytoplasm. The diameter of the field of measurement in the microspectrophotometer was 1.6/tin. In some locations, the rim of the cytoplasm was 553
554
H. PAKKINBI.:RGand R. Fo¢; +
+
+
-I-
10-
5--
g
g
8
~
E
Frontal I(~e 0
Tempoea{ lobe
F'clrleto{ lobe
Occ ~ollat lobe
H
4
-I-
L~
-F
-
4-
10"
izz
I ~ c
~
in the nuclei in the 2 regions are ahnost identical. On the other hand, the nuclear diameter of pyramidal cells in the hippocampus and the thalamus are ver? similar, but gram counts are significantly lower in the hippocampus than in the thalamus. Uridine uptake is quite similar in all parts of the cortex, although slightly increased grain counts are noted in the third and fifth layers. In Table 1 we have compared the grain counts fiom this investigation on the monkey with earlier investigations on mice (Fog and Pakkenberg, 1976). The numbers are c o m p a r a b l e because of a similar exposure time and nucleus area. The monke}, however. was injected with a 200o lower dose of uridine per kg. Striking differences are observed in the second layer of the cortex, the Purkinje cells and the hippocampus cells, all other counts being almost identical. In Table 2 considerable differences in RNA concentration in the cytoplasm in the different brain regions are demonstrated. In each region the extinction value of the different cells has been compared with the grain count of the cell nucleus. From Table 2 it is seen that the correlation coefficient in all cases is negative, and in 8 out of 12 cases significantly different from zero. This indicates that the highest grain counts have been found in cells with a low RNA concentration.
J
Fig. 1. The number of grains in nerve ccll nuclei in the different regions of a grivet brain and in liver and kidney are shown l h after H3-5-uridine injection, the standard error is indicated at the top of each column. too narrow for extinction measurements to be made, e.g. in the dentate gyrus. In the cortical sections, only the nerve cells in the fifth layer were measured. RESULTS As illustrated in Fig. l, the uridine uptake varies from location to location in the brain. The grain counts are given per area, and are therefore comparable from region to region. It is noteworthy that there is no correlation between grain counts and the nuclear diameters in different regions. The diameter of the nuclei in the corpus striatum, e.g. is 11.5 # m as against 18.9 l~m in the cervical cord. The grain counts
DISCUSSION
Regional uridine uptake It is well-established that uridine is especially incorporated in RNA in the cell nucleus (Guroff, Hogans and Udenfriend, 1968). By the autoradiographical method, the small, easily soluble molecules are removed from the tissues by the histological procedure. Only large molecules are fixed. There is no direct correlation between the uridine uptake and the synthesis in the cells (Dunn and Bondy, 1974). However, it is reasonable to assume that the uridine uptake expresses the RNA synthesis, if the animal behaves
Table 1. A comparison between grains covering nerve cell nuclei in the mouse (Fog and Pakkenberg, 1976} and the grivet (grains/'100 jtm 2)
Cortex. 2nd layer Cortex, 5th layer Purkinje cells Striatum Hippocampus, pyr. layer Hippocampus, dentate gyrus Thalamus S.E. in brackets.
Mouse
Monkey
24.4 ( + 2.05) 10.8 ( ± 1.10t 22.0 ( + 3.181 11.4 ( ± 1.581 17.6 ( + 2.18t 7.0 (±0.98) 15.8 ( ± 1.491
9.8 (±0.55) 12.1 (_+0.49) 9.4 ( ± 0.501 I 1.6 ( +0.501 8.6 ( ± 0.401 8.1 ( +_0.53t 16.0 ( ± 0.511
Uridine uptake by nerve cells of the grivet monkey
555
Table 2. The extinction values of the nerve cell cytoplasm in 25 cells in different areas of a grivet brain; staining method: gallocyanine--chrome alum Extinction Frontal cortex Parietal cortex Temporal cortex Occipital cortex Striatum Thalamus Hippocampus (pyr. layer) Substantia nigra Facial nucleus Purkinje cells Spinal cord cervical Spinal cord lumbar
0.32 0.36 0.27 0.30 0.30 0.23 0.34 0.22 0.34 0.38 0.44 0.30
Range 0.22-0.42 0.28-0.46 0.21-0.36 0.18 0.40 0.22 0.39 0.15--0.36 0.23 0.43 0.10-0.39 0.19 0.54 0.28 0 . 5 0 0.3~0.55 0.21 -0.42
Correlation coefficient -0.22 - 0.02 -0.81"** -0.70*** -0.38 - 0.46* -0.67** -0.44* -0.59* -0.83*** -0.05 -0.44*
The correlation coefficients show the relation between uridine labelling and extinction values; the difference from zero is shown. * P < 0.05. ** P < 0.01. *** P < 0.001. normally during the experimental period (Rainbow, 1979). Thus, it is possible to obtain an expression of the metabolic activity (RNA synthesis) in a short period of time. The labelling of cell nuclei in mice is maximal ½h after uridine injection and remains unchanged during the following 6-12 h (Pakkenberg and Fog, 1972). Uridine labelling of RNA will label all RNA species such as ribosomal, transfer and messenger RNA. However, the amount of ribosomal and transfer RNA is rather stable and the estimation that messenger RNA is the principal class of RNA the metabolism of which is fast and can therefore be followed by the technique employed, appears probable. This assumption is supported by the observation, that the turnover time of ribosomal RNA is about 12 days (Von Hungen et al., 1968), but only 30min to 2 h for messenger RNA (Kimberlin, 1967). Our present results indicate that important regional differences in RNA synthesis exist in the normal grivet. Furthermore, in some regions (hippocampus, cerebellum and the second layer of the cortex) differences exist between grivet and mice (Fog and Pakkenberg, 1976). Thus it is confirmed that it is not possible to extrapolate conclusions concerning the uridine uptake (RNA synthesis) from one animal species to another. In order to investigate whether the uridine uptake is related to morphological, biochemical or physiological differences in the different brain areas, we compared our uridine findings with the capillary density in different brain areas (Campbell, 1939). No corr~c~ 5 / 5
D
relation was found. The protein concentration in the cat brain varies only very little from region to region (Gervas-Camacho, Baljagon and De Feudis, 1970). The variation of leucine uptake in the different brain areas in rats is quite different to that of uridine uptake (Altman, 1963). The variations of glucose uptake and cerebral blood flow are similar from region to region, but are different to the uridine uptake pattern (Schwartz and Sharp, 1978; Sokoloff, 1981); it has thus not been possible to find correlations between uridine uptake and other relevant parameters. Uridine labelling and cytoplasmic R N A concentration
Observations on the relationship between cell activity and cytoplasmic RNA concentration are conflicting. For example, Pevzner (1965), Peterson and Erulkar (1973) and Peterson and Kernell (1970) noted a relation between increased cytoplasmic RNA concentration and increased cell activity. On the other hand Bocharova et al. (1972) found an initial decrease in cytoplasmic R N A concentration with an increase after 40 and 7 0 m i n (150-200~o). In vitro investigations (Orrego, 1967) demonstrated a 4 0 ~ decrease in R N A synthesis after electrical stimulation; in contrast, Edstr6m and G r a m p p (1965) and G r a m p p and Edstr6m (1963) claimed that electric stimulation had no bearing on the R N A concentration. In patients who died in status epilepticus, a severe decrease of the R N A concentration in the cytoplasms has been found (Einarson and Krogh, 1955). Electrically induced convulsions (Hartelius,
556
H. PAKKENBERGand R. FoG
1952) in cats have induced foci o f c h r o m o p h o b i c cells (cells with a low cytoplasmic R N A concentration) in the cerebral cortex. The relation between cytoplasmic basophilia and uridine uptake in the nucleus was investigated by Koenig (1958). He found that chromophobic nerve cells incorporate more RNA precursor than chromophilic neurons. Engel and Morrell (1970) observed no correlation between the degree o f chromophilia and the grain counts in normal neurons. We have demonstrated a relation between low RNA concentration in the cytoplasm and high uridine labelling of the nucleus. This might be an expression of an increased activity of these neurons. Earlier we showed that the number of grains in the cytoplasm increases about 6 h after the uridine injection (Pakkenberg and Fog, 1972), but an investigation of the uridine labelling in the nucleus and a quantitative determination of RNA concentration in the cytoplasm in the same cell, has not been previously made. The grain counts in the choroid plexus are of the same order of magnitude as in the most active regions in the brain. In the mouse (Pakkenberg and Fog, 1977) we found a much higher uptake in the plexus than in the nerve cells in the cortex. The relatively modest uptake in the hepatocytes is striking. In the mouse the uptake in the liver is 100~o higher than in the nerve cells (Pakkenberg and Fog, 1977). We have no explanation of these differences.
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